Marker for stem cells and its use

ABSTRACT

A marker for mesenchymal stem cells (MSC) is provided, comprising an integrin alpha10 chain and/or an integrin alpha11 chain expressed on the cell surface of or intracellular in a MSC. The marker is used in methods for identification of mammalian MSC and in methods for isolation of MSC. Also included are isolated cellular populations of mammalian MSC and a cellular composition comprising the latter. Moreover, uses of said marker for isolation, modulation and identification of mammalian MSC are provided.

This is a divisional application of U.S. application Ser. No.10/517,210, filed Mar. 9, 2005, now U.S. Pat. No. 7,749,710, issued Jul.6, 2010, which is a National Stage Entry of PCT InternationalApplication No. PCT/SE03/00983, filed Jun. 12, 2003, which claims thebenefit of priority of Swedish Application No. 0201831-5, filed Jun. 14,2002, and of U.S. Provisional Application No. 60/388,298, filed Jun. 14,2002. All of the above listed applications are hereby incorporated byreference in their entirety.

TECHNICAL FIELD

This invention relates to a marker for isolation and identification ofmammalian mesenchymal stem cells. Also included are methods and uses ofsuch a marker as well as an enriched cellular population and a cellularcomposition comprising the enriched cellular composition.

BACKGROUND OF THE INVENTION

Mesenchymal Stem Cells

The adult body houses so called stem cells that are capable of dividingmany times while also giving rise to daughter cells with specificphenotypical characteristics. Several types of stem cells exist in thebody including embryonic stem cells, haematopoietic stem cells andmesenchymal stem cells. Mesenchymal stem cells are able to formmesenchymal tissues such as bone, cartilage, muscle, bone, ligament, fatand bone marrow stroma. FIG. 1 shows a schedule of suggested stepwisetransitions from putative mesenchymal stem cells (MSC) to highlydifferentiated phenotypes. The mesenchymal stem cells are located inbone marrow, around blood vessels, in fat, skin, muscle, bone and othertissues. Their presence contributes to the reparative capacity of thesetissues.

Medical use of MSC

Currently, the medical use of MSC is to explore their potential in theregeneration of tissues that the body cannot naturally repair orregenerate when challenged. For this, MSC, are isolated, expanded inculture and stimulated to differentiate into connective tissues such asbone, cartilage, muscle, bone marrow stroma, tendon, fat and others.These tissue-engineered constructs can then be re-introduced into thehuman body to repair lost or damaged tissue. In another approach MSC canbe directly stimulated in vivo to induce the formation of specifictissues in situ.

Having defined MSC as potential “building blocks” for tissue engineeringand transplantation, researchers are now searching for better ways toidentify, isolate and characterize MSC.

Alpha10

A newly discovered collagen-binding integrin, alpha10beta1, includes theintegrin subunit alpha10 (Camper et al., (1998) J. Biol. Chem.273:20383-20389). The integrin is expressed on chondrocytes and shows aM_(r) of 160 kDa after reduction when isolated from bovine chondrocytesby collagen type II affinity purification.

Cloning and cDNA sequencing showed that it shares the general structureof other integrin alpha subunits. The predicted amino acid sequenceconsists of a 1167-amino acid mature protein, including a signal peptide(22 amino acids), a long extracellular domain (1098 amino acids) atransmembrane domain (22 amino acids), and a short cytoplasmic domain(22 amino acids). In contrast to most alpha-integrin subunits, thecytoplasmic domain of alpha10 does not contain the conserved sequenceKXGFF(R/K)R (SEQ ID NO: 1). Instead, the predicted amino acid sequencein alpha10 is KLGFFAH (SEQ ID NO: 2). It is suggested that the GFFKR(SEQ ID NO: 3) motif in alpha-chains are important for association ofintegrin subunits and for transport of the integrin to the plasmamembrane (De Melker et al. (1997) Biochem. J. 328:529-537).

The extracellular part contains a 7-fold repeated sequence, an I-domain(199 amino acids) and three putative divalent cation-binding site.Sequence analysis has revealed that the alpha10 subunit is most closelyrelated to the I domain-containing α subunits with the highest identityto alpha1 (37%), alpha2 (35%) and alpha11 (42%).

Alpha11

The alpha11 integrin has recently been identified and cloning andcharacterisation revealed an I-domain containing, beta1-associatedintegrin.

The open reading frame of the cDNA encodes a precursor of 1188 aminoacids. The predicted mature protein of 1166 amino acids contains 7conserved FGGAP (SEQ ID NO: 4) repeats, an I-domain with a MIDAS (SEQ IDNO: 5) motif, a short transmembrane region and a unique cytoplasmicdomain of 24 amino acids containing the sequence GFFRS (SEQ ID NO: 6).

Alpha11 contains three potential divalent cation binding sites inrepeats 5-7. The presence of 22 inserted amino acids in theextracellular stalk portion (amino acids 804-826) distinguishes thealpha11 integrin sequence further from other integrin alpha-chains.

Amino acid sequence comparisons reveal the highest identity (42%) withthe alpha10 integrin chain. Immunoprecipitation with antibodies to thealpha11 integrin captured a 145 kDa protein, distinctly larger than the140 kDa alpha2 integrin chain when analysed by SDS-PAGE undernon-reducing conditions.

Isolation and Identification of MSC

The identification of MSC in situ is hampered by the fact thatmono-specific and unique molecular probes do not exist. It is thereforenecessary to further characterize mesenchymal stem cells to identifyprobes or combinations of probes that can unequivocally identifymesenchymal stem cells in tissue. Such markers will also be useful forthe isolation of mesenchymal stem cells from bone marrow (BM) and bloodtissues.

Approximately one cell out of 10.000-100.000 nucleated cells in bonemarrow aspirates is expected to be a mesenchymal stem cell. Currently,the main method for the isolation of mesenchymal stem cells from bonemarrow is based on their capacity to adhere to plastic culture dishesand form colonies while the majority of bone marrow cells do not adhereand form colonies. These colonies are then further expanded and theninduced with defined factors to differentiate into specific mesenchymaltissues. It is not clear, however, whether the mesenchymal stem cellsisolated this way are a homogenous population. It will therefore beimportant to find markers that can be used to identify subclasses ofmesenchymal stem cells with specific differentiation potentials.

In U.S. Pat. No. 6,200,606, the isolation of cartilage or bone precursorcells from haematopoietic and non-haematopoietic cells by the use ofCD34 as a negative selection marker and the further use of isolated stemcells in bone and cartilage regeneration processes is described. Still,no specific marker for mesenchymal stem cells is identified nordisclosed. The CD34 marker is expressed on early lymphohaematopoieticstem and progenitor cells, small-vessel endothelial cells, embryonicfibroblasts, and some cells in foetal and adult nervous tissue,haematopoietic progenitors derived from foetal yolk sac, embryonicliver, and extra-hepatic embryonic tissues including aorta-associatedhaematopoietic progenitors in the 5 week human embryo.

Pittenger at al. ((1999) Science 284:143-147) have used a densitycentrifugation of human bone marrow to isolate human MSC. Cellularmarkers used to identify the MSC are SH-2, SH-3, CD29, CD44, CD71, CD90,CD106, CD120a, CD124.

Majumdar et al., ((2000) J. Cell. Physiol. 185:98-106) have used CD105as a marker for enrichment of human MSC from bone marrow.

Denni et al., ((2002) Cells Tissues Organs 170:73-82) have used a markercalled Stro-1 to enrich human MSC from bone marrow.

All markers mentioned so far may be used for enrichment of hMSC. Still,they are not exclusive for MSC, the isolated population is heterogenouswhen enriched using these markers. Monospecific and unique probes forthe identification of hMSC do not exist as of today.

Furthermore, markers are needed to monitor the differentiation ofmesenchymal stem cells into specific types of mesenchymal cells. Thiswill be especially important when these cells are re-introduced into thehuman body to replace loss of damaged mesenchymal tissue, such as boneor cartilage.

Finally, the identification of specific cell surface markers formesenchymal stem cells may be used for their isolation out of a complexmixture of cells by cell sorting techniques such as fluorescenceactivated cell sorting (FACS).

It is thus highly desirable in the light of the aforementioned problemsto identify and isolate MSC, for further use in bone, cartilage, muscle,bone marrow, tendon or connective tissue repair in vivo or in vitro. Inthis respect, the present invention addresses this needs and interest.

SUMMARY OF THE INVENTION

In view of the foregoing disadvantages known in the art when trying toisolate and identify mammalian MSC, the present invention providesmarker for mammalian MSC suitable for identifying and isolatingmammalian MSC.

One object with the present invention is to provide methods foridentifying, or isolating mammalian MSC, or an enriched cellularpopulation of MSC.

Another object is to provide uses of the marker according to theinvention for identifying or isolating mammalian MSC.

Thus, the present invention provides a marker for mammalian mesenchymalstem cells. The marker comprises an integrin alpha 10 chain and/orintegrin alpha 11 chain expressed on the cell surface of mesenchymalstem cell or intracellular in a mesenchymal stem cell.

Further embodiments include wherein the integrin alpha10 and/or integrinalpha 11 chain is expressed as a heterodimer in combination with anintegrin beta1 chain.

Also, the present invention provides a method for identifying amammalian mesenchymal stem cell. Such a method comprises the steps of

-   -   a) providing a sample comprising a mesenchymal stem cell,    -   b) detecting integrin alpha10 and/or alpha11 chain expression on        the cell surface of a mesenchymal stem cell or intracellular in        a mesenchymal stem cell,    -   c) scoring the integrin alpha10 and/or alpha11 chain expression,        and    -   d) identifying the mesenchymal stem cell according to the        scoring in c) above.

Further embodiments include wherein the expression in b) above isdetected by detecting the integrin alpha10 and/or integrin alpha 11protein expression.

Even further embodiments include wherein the expression in b) above isdetected by detecting the integrin alpha10 and/or integrin alpha 11 mRNAexpression.

Even further, the present invention provides a method for determiningwhether a test compound modulates a mammalian mesenchymal stem celldifferentiation.

Such a method comprises the steps of

-   -   a) providing a mesenchymal stem cell    -   b) contacting the mesenchymal stem cell with a test compound,        and    -   c) detecting a change in rate or pattern of differentiation of        the mesenchymal stem cell as an indication of that the test        compound modulates mesenchymal stem cell differentiation.

Still even further, the present invention provides a method forproducing an isolated population of mammalian cells enriched formesenchymal stem cells relative a reference population. Such a methodcomprises the steps of

-   -   a) providing at least a portion of a population of cells, or a        portion of a reference population, comprising MSC and at least        one cell other than the mesenchymal stem cells,    -   b) introducing into the population of cells in a) above a        compound identifying the mesenchymal stem cells,    -   c) selecting and isolating from the population of cells in b)        above the mesenchymal stem cells, thereby producing a population        of cells enriched for mesenchymal stem cells.

The method according to the invention may in further embodiments includewherein the mesenchymal stem cells is identified as a mesenchymal stemcell by detecting expression of integrin alpha10 and/or alpha11 chainexpression on the cell surface of said mesenchymal stem cells accordingto the method disclosed in the present invention.

Even further, an enriched mammalian cellular population of mesenchymalstem cells, comprising at least one intact, viable mesenchymal stem cellis disclosed. Such enriched cellular population is a population whereinthe mesenchymal stem cell is characterised by

-   -   a) expressing an integrin alpha 10 chain and/or integrin alpha        11 chain on the cell surface of or intracellular in said        mesenchymal stem cell,    -   b) being substantially free from expression of molecules        specific for committed lymphohaematopoietic cells or uncommitted        stem cells.

Also, an isolated mammalian mesenchymal stem cell expressing a markeraccording to the invention, obtainable by the method for producing apopulation of cells enriched for mesenchymal stem cells according to theinvention is disclosed.

Still even further, a mammalian cellular composition comprising theenriched cellular population according to the invention, or the isolatedmesenchymal stem cell according to the invention is disclosed.

Uses of a marker according to the invention for identification of amammalian mesenchymal stem cell, for modulating differentiation of amammalian mesenchymal stem cell and for isolating a mammalianmesenchymal stem cell are also provided.

SHORT DESCRIPTION OF DRAWINGS

FIG. 1 shows a schematic view of a suggested stepwise transition fromputative mesenchymal stem cell (MSC) to highly differentiatedphenotypes. (From Caplan A. I. and Bruder S. P Trends Mol. Med. 2001,7(6): 259-264).

FIG. 2 shows that human mesenchymal stem cells in culture express bothintegrin alpha10 and alpha11 chains on their cell surface. In thefigure, the upper band in both lanes is alpha10 (in the left lane) andalpha11 (in the right lane). The lower band in both lanes represents thebeta1 chain.

FIG. 3 shows histograms after flow cytometry analysis. Shown in FIG. 3Ethe antibody against alpha10 bound to the HEK293 cells transfected withhuman alpha10 integrin-subunit is seen. The antibody against alpha10 didnot bind to HEK293 cells tranfected with human alpha11 integrin-subunit,as shown in the middle panel (right, FIG. 3F), or untranfected HEK293cells, as shown in FIG. 3D). Shown in FIG. 3I, the antibody againstalpha11 bound to the HEK293 cells transfected with human alpha11integrin-subunit. The antibody against alpha11 did not bind to HEK293cells transfected with human alpha10 integrin-subunit, as shown in FIG.3H, or untransfected HEK293 cells, as shown in FIG. 3G). FIG. 3A-Crepresent control (secondary antibody alone), which did not bind to anyof the HEK293 cells tested.

FIG. 4 shows flow cytometry histograms. After 2 weeks treatment withFGF-2, 96% of the cells treated with FGF-2 expressed the integrinalpha10 (lower panel, FIG. 4 b). Control (secondary antibody alone) isshown in the upper panel in FIG. 4 a.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the terms “rodent” and “rodents” refer to all members ofthe phylogenetic order Rodentia.

The term “murine” refers to any and all members of the family Muridae,including rats and mice.

The term “substantially free from” is herein intended to mean belowdetection limits of the assay used thereby appearing negative, i.e. freefrom.

The term “committed” is herein intended to mean dedicated to, or focusedon. Thus, a committed cell is a cell that is dedicated to, or focused ona specific differentiation pathway. From this it will follow that anuncommitted cell is not dedicated to, or focused on, any specificdifferentiation pathway and has several options.

Integrin Alpha10 and Integrin Alpha11 as a Marker for MSC

We have surprisingly found that the integrins alpha10beta1 andalpha11beta1 are present on human mesenchymal stem cells. Thus, theseintegrins can be used to identify, differentiate, and isolatemesenchymal stem cells from a mixed cell population and will be a usefultool in cell therapy to repair damaged tissue.

The human integrin alpha10 chain sequence is known and publiclyavailable at GenBank™/EBI Data Bank accession number AF074015. Thus, newuses and methods of the integrin alpha10 chain are disclosed in thepresent invention.

The human integrin alpha11 chain sequence is known and publiclyavailable at GenBank™/EBI Data Bank accession number AF137378. Thus, newuses and methods of the integrin alpha11 chain are disclosed in thepresent invention.

As revealed above, the present invention relates to a marker formesenchymal stem cells (MSC), comprising an integrin alpha 10 chainand/or integrin alpha 11 chain expressed on the cell surface ofmammalian MSC or intracellular in mammalian MSC.

In a further embodiment, the integrin alpha10 and/or integrin alpha 11chain is expressed as a heterodimer in combination with an integrinbeta1 chain.

Mammalian MSC is generally isolated from bone marrow, peripheral blood,cord blood, liver, bone, cartilage, perichondrium, muscle, periosteum,synovium or fat. The isolation may be based on the cells capacity toadhere to plastic culture dishes and form colonies under specificculture conditions, while the majority of bone marrow cells do notadhere and form colonies. Suitable protocol for isolation of mammalianMSC, without including the marker according to the invention, is furthergiven in detail in Mason J M et al (2000, Cartilage and boneregeneration using gene-enhanced tissue engineering. Clin. Orthop.379S:S171-178), Chu C R et al (1997, Osteochondral repair usingperichondrial cells in Clin. Orthop. 340:220-229 (2000), and Dounchis JS et al (2000, Cartilage repair with autogenic perichondrium cell andpolylactic acid grafts. Clin. Orthop. 377:248-264). Thus, known methodsmay be a used, but with the introduction of the marker(s) according tothe invention.

The colonies may further be expanded and then induced with definedfactors to differentiate into specific mesenchymal tissues. Forchondrocytes, the culture is a culture in pelleted micromass or inalginate without serum, and with TGFbeta3 added as a defined factor. Forosteogenic cells, cells may be cultured in the presence ofdexamethasone, beta-glycerol phosphate, ascorbate, and 10% FBS (foetalbovine serum), and for adipocytes, cells may be cultured in the presenceof 1-methyl-3-ispbutylxanthine, dexamethasone, insulin, andindomethacin.

Thus, the use of the marker(s) according to the invention in theisolation and expansion protocols will give a homogenous MSC population.Mesenchymal stem cells are not isolated and expanded this way are ahomogenous population. More details concerning suitable factors to beincluded in cutures for expansion of specific MSC are given by Caplan, AI (1991, Mesenchymal Stem Cells. J. Orthop. Res. 9:641-650), Pittenger MF et al. (1999, Mutlilineage potential of adult human mesenchymal stemcells, Science. 284:143-7), and by Minguell J J, Erices, A and Conget, P(2001, Mesenchymal Stem Cells. Exp. Biol. Med. 226(6):507-520 —table ofall factors that may be required to differentiate cells) all referencesare incorporated herein by reference.

Human MSC may be isolated from bone marrow, peripheral blood, cordblood, liver, bone, cartilage, perichondrium, muscle, periosteum,synovium or fat. The MSC may then further be isolated following adensity centrifugation and found as a part of a mononuclear cellfraction layer at the density interface of 1.073 g/ml (Percoll™,Pharmacia). Suitable protocols are given in detail in Vogel W et al(2003, Heterogeneity among bone marrow-derived mesenchymal stem cellsand neural progenitor cells) and by Nevo, Z et al (1998, The manipulatedmesenchymal stem cells in regenerated skeletal tissues. Cell Transplant7:63-70) both references incorporated herein by reference.

Out of this mononuclear cell fraction, 1/10 000- 1/100 000 cells formcolonies upon culture in serum in culture dishes (Bruder S P et al(1997) Growth kinetics, self-renewal, and the osteogenic potential ofpurified human mesenchymal stem cells during extensive subcultivationand following cryopreservation. J. Cell. Biochem. 64:278-294).

Thus, including the marker according to the invention, comprising anintegrin alpha 10 chain and/or integrin alpha 11 chain in knownisolation and expansion protocols, as well as using the marker(s) alone,will be highly valuable for further evaluation and enrichment of the MSCpopulation. Particularly, no other specific and unique marker as themarker according to the invention for mammalian MSC is known.

A Method for Identifying MSC

According to the invention, a method for identifying a mammalian MSC isdisclosed. The method comprises the steps of

-   -   a) providing a sample comprising MSC,    -   b) detecting integrin chain alpha10 and/or alpha11 expression on        the cell surface of a MSC or intracellular in MSC,    -   c) scoring the integrin alpha10 and/or alpha11 chain expression,        and    -   d) identifying the MSC according to the scoring in c) above.

In more detail, the method according to the invention may furthercomprise the steps of:

-   -   e) providing a cell suspension comprising mammalian mesenchymal        stem cells,    -   f) contacting the cell suspension in e) with a monoclonal        antibody or fragments thereof binding to the integrin        alpha10beta1/or alpha11beta1, under conditions wherein said        monoclonal antibody or fragments thereof form an        antibody-antigen complex with the extracellular domain of        integrin alpha10beta1/alpha11beta1,    -   g) separating cells binding to said monoclonal antibody or        fragments thereof in f), and optionally    -   h) recovering the cells binding to the monoclonal antibody or        fragments thereof in g) from said antibody or fragments thereof,

thereby producing a population of mammalian mesenchymal stem cells,optionally free from said antibody or fragments thereof.

The cell suspension provided in e) above, comprising mammalian MSCs maybe isolated from bone marrow, peripheral blood, cord blood, liver, bone,cartilage, muscle, perichondrium, periosteum, synovial tissue, fat orany tissue comprising MSCs. The cell suspension may further be isolatedfrom mammalian iliac crest, femora, tibiae, spine, rib or othermedullary spaces. Other sources of human MSCs include embryonic yolksac, placenta, and umbilical cord. If the population of cells iscollected from BM, only 0.01-0.001% of the starting population, or“crude population”, are MSCs. Though, this may vary between differentdonors.

In one further embodiment, the mammalian MSCs are human MSCs.

In one further embodiment, the mammalian MSCs are murine MSCs.

In one further embodiment, the culture above is a culture for 2-4 weeks.

In one embodiment, the method for isolating a population of MSCs furthercomprises the steps of

-   -   i) collecting bone marrow aspirate (5-30 ml) from a human        patient into a syringe containing e.g. heparin to prevent        clotting,    -   j) washing the marrow sample with e.g. Dulbecco's        phosphate-buffered saline (DPBS) or any similar saline solution,        and recovering the cells after centrifugation at 900 g, and        repeating this procedure once more.    -   k) loading the cells onto 25 ml of Percoll of a density of 1.073        g/ml in a 50-ml conical tube and separating the cells by        centrifugation at 1100 g for 30 min at 20° C.,    -   l) collecting the nucleated cells from the interface, diluting        with two volumes of DPBS, and collecting by centrifugation at        900 g. Resuspending the cells counting the cells, and plating        out the cells at the required density, suitable        200,000-cells/cm²,    -   m) culturing the cells in Dulbecco's modified Eagle's medium        (DMEM) or any other suitable medium (low glucose) containing 10%        foetal bovine serum (FBS),    -   n) replacing the medium at 24 and 72 hours and every third or        fourth day thereafter, and    -   o) subculturing the hMSCs that grow as symmetric colonies at 10        to 14 days by treatment with 0.05% trypsin and 0.53 mM EDTA for        5 min, rinsed from the substrate with serum-containing medium,        collected by centrifugation at 800 g for 5 min, and seeded into        fresh flasks at 5000 to 6000 cells/cm².

The separation of MSCs is a selection and isolation step for separatingthe identified MSCs. Various techniques known to the skilled artisan maybe employed to separate the cells by initially removing cells dedicatedto other lineages than MSCs.

If an antibody or fragments thereof is used it may be attached to asolid support to allow for a highly specific separation. The particularprocedure for separation employed, e.g. centrifugation, mechanicalseparation, such as columns, membranes or magnetic separation, shouldmaximize the viability of the fraction to be collected. Varioustechniques of different efficacy may be employed known to a personskilled in the art. The particular technique employed will depend uponefficiency of separation, cytotoxicity of the methodology, ease andspeed of performance, and necessity for sophisticated equipment and/ortechnical skill.

Procedures for separation of MSCs from a cell suspension aided by themethod according to the invention may include magnetic separation, usinge.g. antibody-coated magnetic beads, affinity chromatography based onthe antibody or fragments thereof according to the invention, and“panning” with an antibody or fragments thereof attached to a solidmatrix, e.g., a plate, or other convenient techniques.

Techniques providing accurate separation include fluorescence activatedcell sorters by the use of e.g. an antibody or fragments thereof in themethod according to the invention, which can have varying degrees ofsophistication, e.g., a plurality of colour channels, light scatteringdetecting channels, impedance channels, etc. known to the skilled man inthe art.

In one embodiment, a first enrichment step of MSCs in the provided cellpopulation is made. This first selection may be a negative selection ofthe MSCs, i.e. other lineage-committed cells are depleted, or removed,from the initial population of cells.

In still a further embodiment, the first enrichment is a positiveselection of MSCs that may be repeated until the desired purity of theMSCs is achieved.

As described in the paragraph above the MSC may be isolated by plasticadhesion of a mixed cell population, followed by further optionalexpansion of the cells with defined factors to differentiate intodifferent mesenchymal tissues. For chondrocytes, the culture may be aculture in pelleted micromass or in alginate with or without serum, andwith TGFbeta3 added as a defined factor. For osteogenic cells, cells maybe cultured in the presence of dexamethasone, beta-glycerol phosphate,ascorbate, and 10% FBS (foetal bovine serum), and for adipocytes, cellsmay be cultured in the presence of 1-methyl-3-ispbutylxanthine,dexamethasone, insulin, and indomethacin. More suitable factors areexemplified by Minguell J J, Erices, A and Conget, P (2001) inMesenchymal Stem Cells. Exp. Biol. Med. 226(6):507-520 incorporatedherein by reference.

In further embodiments of the invention, other less specific andnon-unique mammalian MSC markers may be analysed in parallel with themarker according to the invention. Such other markers are SH-2, SH-3,CD29, CD44, CD71, CD90, CD106, CD120a, CD124, CD105, and Stro-1 that MSCmay express. Though, these markers are not unique for mammalian MSC.Markers that do not express on MSC are CD14, CD34 and CD45 and theirexpression, or lack of expression, may in further embodiments also beevaluated in the method according to the invention.

In a further embodiment, the expression above is detected by detectingthe integrin alpha10 and/or integrin alpha 11 protein expression.

The expression of alpha10/alpha 11 may in one embodiment be analysed byfluorescent cell sorting, by using e.g. a fluorescence activated cellsorter (FACS®) or any other methodology having high specificity.Multi-colour analyses may be employed with the FACS, which isparticularly convenient. MSCs may, thus, be separated on the basis ofthe level of staining for the particular antigens.

In a first separation, antibodies for other markers may be used labelledwith one or more fluorochrome(s). Other markers to be used may infurther embodiments be SH-2, SH-3, CD29, CD44, CD71, CD90, CD106,CD120a, CD124, CD105, and Stro-1 that MSCs may express. Markers that arenot expressed on MSCs are CD14, CD34 and CD45 and their expression, orlack of, may in further embodiments also be evaluated in the methodaccording to the invention or a fragment.

If further lineages or cell populations not being MSCs are to be removedin one step, various antibodies to such lineage-specific markers may beincluded.

Fluorochromes, which may find use in a multi-colour analysis, includephycobiliproteins, e.g., phycoerythrin and allophycocyanins,fluorescein, Texas red, etc. well known to the skilled man in the art.

The MSCs may be selected against dead cells, by employing dyesassociated with dead cells (propidium iodide, LDS). The cells may becollected in a medium comprising foetal calf serum.

MSCs may as well be selected based on light-scatter properties and theirexpression of various cell surface antigens, in combination with theidentification using the method according to the invention.

Alternatively, MSCs may be analysed by immunoprecipitation therebydetecting and identifying integrin alpha10 and/or alpha11 chainexpression. A suitable immunoprecipitation protocol is given in briefbelow. Any other suitable immunoprecipitation method may, of course, beused in the method according to the invention.

In brief,

-   -   1. Antibodies against the cytoplasmic domains of integrin        subunits alpha10 and alpha11 can be used to specifically        immunoprecipitate integrins alpha10beta1 and alpha11beta1        respectively from cell lysates. Polyclonal antibodies suitable        for immunoprecipitation of integrin alpha10 or alpha11 are known        and published in J. Biol. Chem. (1998, 273: 20383-9).    -   2. MSCs expressing either the integrin subunit alpha10 or        alpha11 may then be grown in a suitable cell culture medium e.g.        DMEM, IMEM, RPMI optionally with serum and growth factors. Cells        adherent on the plate are washed once with PBS and then surface        biotinylated using e.g. 0.5 mg/ml Sulfo-NHS-LC-biotin (Pierce)        in 4 ml PBS for 20 min on ice or any other suitable        biotinylation reagent known to a man skilled in the art.    -   3. Cells are then washed once with PBS and 10 ml 0.1M        glycine/PBS were added for 5 min on ice. After washing once with        PBS cells are lysed in 1 ml lysis buffer (1% NP-40, 10%        glycerol, 20 mM Tris/HCl, 150 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂,        protease inhibitor cocktail Roche, pH7.5) on ice.    -   4. The cell lysate is spun down at 15.000 g for 10 min and the        supernatant removed and incubated with 1 μl of α10/α11        pre-immune serum and then 20 μl Prot G sepharose (Amersham) in        100 μl lysis buffer is added.    -   5. After rotating 1 h at 4° C. the lysate is centrifuged for 1        min at 8000 rpm and the supernatant is collected. For each        subsequent immunoprecipitation 150 μl cell lysate supernatant is        pipetted into an eppendorf tube and 1 μl of antiserum or        monoclonal antibody solution is added.    -   6. Antibodies used are rabbit-anti-human α10 serum and for        rabbit-anti-human α11 serum (both sera against the cytoplasmic        domains of the integrins published by Tiger et al.,        (Developmental Biology (2001) 237:116) for alpha11 and by Camper        et al (J. Biol. Chem. (2001) 306:107-116) for alpha10).    -   7. After 2 h rotating at 4° C., 20 μl prot G sepharose        (Amersham) in 1000 lysis buffer is added and the mixture further        rotated for another 45 min. The Sepharose-beads are then spun        down briefly and washed three times with lysis buffer.    -   8. 20 μl SDS PAGE sample buffer (including 100 mM DTT) is added        to the sepharose beads and the samples boiled for 5 min.    -   9. 5 μl of each sample is run on an 8% straight gel (Novex) and        then electro-transferred onto a PVDF membrane. The membrane is        blocked in 2% BSA/Tris buffer salin 0.05% Tween for 1 h, washed        once with Tris buffer salin 0.05% Tween and then incubated with        2 μl Extravidin-peroxidase (Sigma) in 8 ml blocking buffer.        After 1 h the Extravidin-peroxidase solution is removed and the        membrane washed 3×20 min in TBST. Surface biotinylated proteins        can then be detected with e.g. ECL (Amersham) and visualised on        a photographic film.

In still a further embodiment, the integrin chain alpha10 and/or alpha11expression is detected on the cell surface of a MSC or intracellular ina MSC in the method according to the invention. Methods given above,e.g. flow cytometry and immunoprecipitation may be used.

In still a further embodiment, the expression in b) above is detected byany immunoassay, such as the methods described in Immunochemicalprotocols (Methods in molecular biology, Humana Press Inc). Thedetection may be performed by various methods, e.g. any immunomethodknown to the skilled man in the art, such as immunoprecipitation,Western blotting or flow cytometry methods, e.g. fluorescence activatedcell sorting (FACS). Antibodies, such as monoclonal antibodies orfragments thereof, are particularly useful for identifying markers,surface membrane proteins as well as intracellular markers, associatedwith particular cell lineages and/or stages of differentiation. Thus, itis suitable for the identification of integrin alpha10 as well asalpha11. Still, identification may as well be performed by any specificmolecule, such as a protein or peptide, binding specifically to theintegrin alpha10 and/or the integrin alpha11 molecule. Examples of suchproteins or peptides are natural ligands, binding to the integrinalpha10 and/or the integrin alpha11 molecule. Such natural ligands maybe made recombinant, chemically synthesised, or purified from a naturalsource. In still a further embodiment, the expression above is detectedby detecting the integrin alpha10 and/or integrin alpha 11 mRNAexpression. Detection of mRNA expression of a specific protein is wellknown to the skilled man in the art, and is generally done by probingthe mRNA with a DNA or RNA probe specific for the mRNA of interest,under hybridisation conditions where the probe is not hybridising toother mRNA molecules. Different polymerase chain reactions (PCR) mayalso be used, which is obvious to the skilled man in the art.

A suitable PCR-method is given below. In brief, polymerase chainreaction (PCR) may be used.

RNA may be prepared from human mesenchymal stem cells or chondrogenicprecursor cells by standard methods, for example by the use of RNeasyMini Kit (Qiagen Germany).

cDNA may be produced by reverse transcriptase reaction, Superscript II(Invitrogen, USA) according to manufacturers recommendation with oligod(T)-primers or gene specific primers.

PCR is thereafter performed to amplify the cDNA. Specific primers forα10, forward 5′ GCT CCA GGA AGG CCC CAT TTG TG 3′ (SEQ ID NO: 7) andreverse 5′GTG TTT TCT TGA AGG GTG CCA TTT 3′ (SEQ ID NO: 8) or for α11,forward 5′GCT GCA GGC AGT GAC AGT A 3′ (SEQ ID NO: 9) and reverse 5′GCGATG GGA ATG GTG ATC T 3′ (SEQ ID NO: 10) are added to the cDNA and thespecific product is amplified by Platinum Taq DNA polymerase(Invitrogen, USA) according to their recommendations at 65° for 30cycles.

The scoring of the integrin alpha10 and/or the integrin alpha11 moleculeexpression may be done relative to a reference cell populationexpressing the integrin alpha10 and/or the integrin alpha11 molecule, aswell as to a cell population not expressing the integrin alpha10 and/orthe integrin alpha11 molecule. Examples of cells expressing the integrinalpha10 and alpha11 are C2C12 cells, HEK293 cells transfected withalpha10 and alpha11 integrin sequences. Examples of cells not expressingalpha10 and alpha11 are non-transfected C2C12 cells and HEK293 cells.

A Method for Producing an Isolated Population of Cells Enriched forMammalian MSC

According to the invention, a method is disclosed for producing anisolated population of cells enriched for mammalian MSC relative areference population, the method comprising the steps of

-   -   a) providing a at least a portion of a population of cells, or        at least a portion of a reference population, comprising MSC and        at least one cell other than a MSC,    -   b) introducing into the population of cells in a) above a        compound identifying the MSC,    -   c) selecting and isolating from the population of cells in b)        above the MSC, thereby producing a population of cells enriched        for MSC.

Providing a population is described in the paragraphs above, and may beperformed in a similar way as in the method for identification of MSC.If the population of cells is collected from BM, at about 0.01-0.001% ofthe starting population, or “crude population”, is MSC. Though, this mayvary between different donors.

The compound introduced to identify the MSC may be a protein, peptide,monoclonal antibody, or part thereof, or polyclonal antibody identifyingthe MSC. In one embodiment, the MSC is identified as a MSC by detectingexpression of integrin chain alpha10 and/or alpha11 expression on thecell surface of said MSC according to the method for identifying MSCdescribed above.

The selection and isolation of MSC is a separation step for separating,and thus isolating, the identified MSC. Various techniques may beemployed to separate the cells by initially removing cells dedicated toother lineages than MSC. Monoclonal or polyclonal antibodies, or partsthereof, are particularly useful for identifying markers, here on intactviable cells, wherein the markers are surface membrane proteinsassociated with particular cell lineages and/or stages ofdifferentiation. The compound used to identify the MSC may also be usedfor the separation step. Thus, said compound(s), such as antibodies, orparts thereof, may be attached to a solid support to allow for a firstcrude separation. Examples of solid supports are beads e.g. magneticbeads, agarose or other similar types of beads known to the skilled manin the art. Any means suitable for separation of cells may be employedon the condition that the separation is not unduly detrimental to theviability of a cell.

The separation techniques employed should maximize the retention ofviability of the fraction to be collected. The assessment of viabilityis described below.

In brief, assessment of cell viability may be performed using e.g. flowcytometry. Thus, after staining cells, but before running on flowcytometer, the following amount/concentration of an appropriate cellviability dye can be added to discriminate between live/dead cells. Anumber of such dyes exist, of which examples and typical methods forusing them are described. The principle is the same for most of thesedyes: these dyes enter the cells if the cell membrane is compromised; assuch, cells that stain with these dyes are dead, and cells that do notstain are considered live.

Examples of dyes are:

a) Propidium Iodide (PI):

-   Stock: 50 ug/uL in ethanol/PBS-   Add: 1 uL per 100 uL of media in tube.    b) 7-Aminoactinomycin D (7 AAD)-   Stock: 1 mg/mL in MeOH-   Add: 1 uL per 100 uL of media in tube    c) To-Pro3-   Stock: 1 mM in DMSO-   Add: 1 uL per 500 uL to 1 mL of solution    d) Ethidium Monoazide (EMA)-   Stock: 50 ug/mL in EtOH-   Add: 5 ug/mL per 1×10⁶ cells

Other methods may be described in Flow Cytometry and Cell Sorting(Springer Lab Manual) by A. Radbruch Springer Verlag (2^(nd) edition,January 2000)

The cell viability of the fraction collected is >90%, preferably 95, 96,97, 98, 99, 99.9, or even 100%.

The particular technique employed for separation of cells in the methodaccording to the invention will depend upon efficiency of separation,cytotoxicity of the methodology, ease and speed of performance, andnecessity for sophisticated equipment and/or technical skill.

Procedures for separation may include magnetic separation, using e.g.antibody-coated magnetic beads, affinity chromatography, cytotoxicagents joined to a monoclonal antibody or used in conjunction with amonoclonal antibody, e.g., complement and cytotoxins, and “panning” withantibody attached to a solid matrix, e.g., a plate, or other convenienttechniques. Techniques providing accurate separation includefluorescence activated cell sorters, which can have varying degrees ofsophistication, e.g., a plurality of colour channels, light scatteringdetecting channels, impedance channels, etc. known to the skilled man inthe art.

Further protocols for separation methods suitable to be used in themethod according to the invention are described by Orfao, A andRuiz-Arguelles, A ((1996) General Concepts about Cell SortingTechniques. Clin Biochem. 29(1):5-9), and by Herzenberg, L A, De Rose, SC and Herzenberg, L A ((2000) Monoclonal Antibodies and FACS:complementary tools for immunobiology and medicine. Immunol. Today.21(8):383-390).

In one embodiment of the method according to the invention, at least oneenrichment step of mammalian MSC is included.

In still a further embodiment, the first enrichment step of MSC is anegative selection of the MSC, i.e. other lineage-committed cells aredepleted, or removed, from the initial population of cells. Examples ofsuch cells to be removed in the negative selection are identified by thefollowing markers: CD14 (monocytes, granulocytes, dendritic cells,macrophages and B cells), CD34 (haematopoietic progenitor cells) andCD45 (leukocytes). These and other markers useful for negative selectionof mammalian MSC are described in detail by Conget, P A, Minguell J J((1999) Phenotypical and functional properties of human bone marrowmesenchymal progenitor cells. J. Cell Physiol. 181:67-73), and byPittenger M F et al. ((1999) Mutlilineage potential of adult humanmesenchymal stem cells. Science. 284:143-7) both references incorporatedherein by reference.

In still a further embodiment, the first enrichment is a positiveselection of MSC that may be repeated till the desired purity of the MSCis achieved. For a positive or a negative selection, proteins, peptides,monoclonal or polyclonal antibodies may be used as a compound toidentify the integrin alpha10 or integrin alpha 11 molecule as describedabove. The compound may be conjugated with means for separation, such asmagnetic beads, which allow for direct separation; biotin, which can beremoved with avidin; or streptavidin bound to a support; fluorochromes,which can be used with a fluorescence activated cell sorter; or thelike, to allow for ease of separation of the particular cell type asexemplified in the paragraphs above. Any technique may be employed whichis not unduly detrimental to the viability of the cells of interest,i.e. the MSC.

In one embodiment, the selection is performed by fluorescent cellsorting, by using e.g. a fluorescence activated cell sorter such as aFACS®, or any other similar methodology having high specificity.Multi-colour analyses may be employed with the FACS which isparticularly convenient and the technique well known to person skilledin the art of flow cytometry. The cells may be separated on the basis ofthe level of staining for the particular antigens. In a firstseparation, antibodies for other markers may be used labelled with onefluorochrome, while the antibodies for the dedicated lineages, i.e. theintegrin alpha10 and/or integrin alpha11, may be conjugated to (a)different fluorochrome(s). Other markers may in further embodiments beSH-2, SH-3, CD29, CD44, CD71, CD90, CD106, CD120a, CD124, CD105, andStro-1 that MSC may express. Markers that are not expressed on MSC areCD14, CD34 and CD45 and their expression, or lack of, may in furtherembodiments also be evaluated together with the marker according to theinvention, e.g. integrin alpha10 and/or integrin alpha11 expression.

If further lineages or cell populations are to be removed in this step,various antibodies to such lineage specific markers may be included.Fluorochromes which may find use in a multi-colour analysis includephycobiliproteins, e.g., phycoerythrin and allophycocyanins,fluorescein, Texas red, etc. The cells may be selected against deadcells, by employing dyes associated with dead cells such as propidiumiodide or LDS-751 (Laser Dye Styryl-751(6-dimethylamino-2-[4-[4-(dimethylamino)phenyl]-1,3-butadienyl]-1-ethylquinolinium perchlorate)). The cells may be collected in any suitablecell culturing media, such as Iscove's modified Dulbecco's medium(IMDM), or in any physiological saline solution, preferably buffered,such as phosphate buffer saline (PBS), optionally with foetal calf serum(FCS) or bovine serum albumin (BSA) present. Other techniques forpositive or negative selection may be employed, which permit accurateseparation, such as affinity columns, and the like, further described bySilvestri F, Wunder E, Sovalat H, Henon P, Serke S in Positive selectionof CD34+ cells: a short review of the immunoadsorption methods currentlyavailable for experimental and clinical use(Report on the “2nd EuropeanWorkshop on stem Cell Methodology”, Mulhouse, France, May 3-7, 1993. J.Hematother. 1993 Winter; 2(4):473-81) and by Basch R S, Berman J W,Lakow E. in Cell separation using positive immunoselective techniques (JImmunol Methods. 1983 Feb. 11;56(3):269-80).

Cells may be selected based on light-scatter properties as well as theirexpression of various cell surface antigens.

While it is believed that the particular order of separation is notcritical to this invention, the order indicated is one way of performingthe invention that is known to work. Thus, suggestively, cells areinitially separated by a crude separation, preferably a negativeselection removing cells not committed for MSC using negative cellmarkers such as CD14, CD34 and CD45. Such cells are negative for theexpression of integrin alpha10 and/or alpha11. The negative selection isfollowed by a fine separation, which is a positive selection, whereinthe positive selection is of a marker associated with MSC and negativeselection for markers associated with lineage committed cells, and otherstem cell populations not being MSC. This separation is then followed byselection for a cellular population, or a cellular compositioncomprising said population, having multi-lineage potential as a MSC andenhanced self-regeneration capability. The composition is furtherdescribed below.

Isolated Mammalian MSC

According to the invention, an enriched cellular population of mammalianMSC is disclosed. Such a cellular population comprises intact, viableMSC, wherein the MSC are characterised by

-   -   a) expressing an integrin alpha 10 chain and/or integrin alpha        11 chain on the cell surface of said MSC or intracellular in        MSC,    -   b) being substantially free from expression of molecules        specific for committed haematopoietic cells.

Molecules specific for committed haematopoietic cells are e.g. CD45.Other molecules the MSC cells are substantially free from are e.g. CD34and CD14.

In further embodiments, the enrichment of such a population is about 70,80, 90, 95, 98, 99, 99.9, or even 100%. The viability of such cells isdiscussed in detail above.

According to the invention an isolated MSC expressing a marker accordingto the invention is disclosed. The isolated MSC are obtainable by themethod for producing a population of cells enriched for MSC according tothe invention.

A Cellular Composition

According to the invention, a mammalian cellular composition isdisclosed. Such a composition comprises the enriched mammalian cellularpopulation according to the invention, or the isolated mammalian MSCaccording to the invention.

Compositions having greater than 90%, usually greater than about 95%,such as 97, 98, 99.9%, of human MSC cells may be achieved according tothe disclosed methods for enrichment of MSC. Such MSC are able toprovide for cell regeneration and development of members of all of thevarious lineages of MSC, such as osteocytes, chondrocytes, e.g.hypertrophic chondrocytes, muscle cells, myotubes, stromal cells, T/Lfibroblasts, adipocytes, tenocytes, dermal cells and other cells. Thisis generally done in cultures, supplied with specific factors describedearlier.

Ultimately, a single cell may be obtained from a MSC composition andused for long-term reconstitution of a mammal deficient for MSC and/ormesenchymal tissue formation or regeneration. The MSC composition shouldbe administered in a therapeutic effective dosage, wherein the dosage isa specific cell number able to repopulate said mammal, such as a humanbeing. This cell number may be different from donor to donor and may bedetermined empirically form case to case by a person skilled in the artof cell transplantation of cells to mammals, such as a human being, inthe need thereof.

Various procedures can be contemplated for transferring and immobilisingthe MSCs, and the composition comprising MSC, including injecting theisolated cells into the site of skeletal defect e.g. damage to articularcartilage, incubating isolated cells in suitable gel and implanting,incubating with bioresorbable scaffold, or by systemically infusing etc.Different procedures are known and described in detail by e.g. Risbud, MV and Sittenger M ((2002) Tissue Engineering: advances in in vitrocartilage regeneration. Trends in Biotech. 20(8):351-356), by Caplan, A.and Bruder, S. P. ((2001) Mesenchymal stem cells: building blocks formolecular medicine in the 21st century. Trends Mol. Med. 7(6):259-64),by Lazarus, H M et al ((1995) Ex vivo expansion and subsequent infusionof human bone marrow-derived stromal progenitor cells (mesenchymalprogenitor cells): Implications for therapeutic use. Bone MarrowTransplant 16:557-564), and by Koc O N et al ((2000) Rapid hematopoieticrecovery after coinfusion of autologous-blood stem cells andculture-expanded marrow mesenchymal stem cells in advanced breast cancerpatients receiving high-dose chemotherapy. J. Clin. Oncol 18(2):307-16).

Optionally MSCs can be incubated with an antibody to the integrinalpha10 or alpha11 in order to hold the cells in place. Thus antibodiescan be conjugated to a bioresorbable scaffold allowing immobilisation ofthe cells before implantation into the damaged or defect site, e.g. intothe site of a skeletal defect. The scaffold allows 3D immobilization ofMSCs. Suitable biomaterial scaffolds are exemplified below. The examplesgiven are not limiting the use of other suitable scaffolds obvious to askilled artisan to choose if more suitable for the particularapplication.

Types of scaffold include, bioresorbable poly(α-hydroxy esters)scaffolds such as polylactic acid (PLLA), polyglycolic acid (PGA) andcopolymer (PLGA).

Further embodiments include scaffolds derived from polymeric gels suchas hyaluronic acid, collagen, alginate and chitosan.

Further embodiments include scaffolds derived from porous carriers, suchas tricalcium phosphate and/or hydroxyapatite ceramic block (Luyten, F.P, Dell'Accio, F and De Bari, C (2001) Skeletal tissue engineering:opportunities and challenges. Best Prac & Res. Clin. Rheum.15(5):759-770.)

The cellular composition according to the invention may be used fortreatment of genetic diseases. Genetic diseases associated with MSC maybe treated by genetic modification of autologous or allogeneic MSC tocorrect the genetic defect. For example, diseases such as differentconnective tissue diseases, e.g. osteogenesis imperfecta, Ehlers Danlossyndrome, Chondrodysplasia, Alport syndrome may be corrected byintroduction of a wild-type gene into the MSC, either by homologous orrandom recombination. Methods for homologous recombination forcorrection of diseases are known and described by Hatada S, Nikkuni K,Bentley S A, Kirby S, Smithies O.((2000) Gene correction inhematopoietic progenitor cells by homologous recombination. Proc NatlAcad Sci USA 97(25):13807-11).

With allogeneic MSC, normal cells form a mammal of the same specieslacking the genetic defect can be used as a therapy. Other embodimentsof gene therapy may be introduction of drug resistance genes to enablenormal MSC to have an advantage and be subject to selective pressure,e.g. the multiple drug resistance gene (MDR). More details are given inAran J M, Pastan I, Gottesman M M (1999) Therapeutic strategiesinvolving the multidrug resistance phenotype: the MDR1 gene as target,chemoprotectant, and selectable marker in gene therapy. (Adv Pharmacol46:1-42).

Diseases other than those associated with MSC may also be treated, wherethe disease is related to the lack of a particular secreted product suchas a hormone, enzyme, interferon, factor, or the like. By employing anappropriate regulatory initiation region, inducible production of thedeficient protein may be achieved, so that production of the proteinwill parallel natural production, even though production will be in adifferent cell type from the cell type that normally produces suchprotein. It is also possible to insert a ribozyme, antisense or othermessage to inhibit particular gene products or susceptibility todiseases, particularly connective tissue

Modulation of MSC

According to the invention a method for determining whether a testcompound modulates a mammalian MSC differentiation is disclosed. Such amethod comprises the steps of

-   -   a) providing a MSC    -   b) contacting the MSC with a test compound, and    -   c) detecting a change in rate or pattern of differentiation of        the MSC as an indication that the test compound modulates MSC        differentiation.

The MSC provided may be an enriched cell population achieved accordingto any of the methods disclosed, the isolated MSC according to theinvention, or the cellular composition according to the invention.

The test compound may be any compound known to affect or suspected toaffect MSC, e.g. pharmaceutical compositions, drugs, polyclonal ormonoclonal antibodies, or parts thereof, such as antibodies binding tointegrin alpha10 and/or integrin alpha11 or any other molecule on theMSC, factors used to promote growth of MSC, e.g. FGF or foeatal bovineserum (FBS), or factors used to promote differentiation of MSC, e.g.dexamethasone, TGFbeta, insulin.

The detection of a change in rate or pattern of e.g. differentiation ofthe MSC as an indication that the test compound modulates MSCdifferentiation may be done via flow cytometry or any other suitablemethod, such as any immunomethod, known to a person skilled in the art.The change in rate or pattern of differentiation may be kinetic,functional or phenotypical studies of the MSC modulated with the testcompound, relative for an untreated, or mock treated, MSC population. Itmay also be a comparison relative to least one second test compound.

In a further embodiment the MSC is identified as a MSC by detectingexpression of integrin chain alpha10 and/or alpha11 expression on thecell surface of said MSC or intracellular in MSC according to the methodof identifying MSC disclosed herein.

Use of the Mammalian MSC

The mammalian MSC, such as human or mouse MSC, provided herein find anumber of uses. For instance, 1) re-generation of a host deficient inMSC; 2) treatment of a host by the re-engraftment of MSC forre-generation of bone, cartilage, muscle, marrow, tendon/ligament andconnective tissue in a patient in the need thereof; 3) in detecting andevaluating growth factors relevant to MSC self re-generation; 4) indevelopment of MSC lineages and screening for factors associated withtheir development and differentiation.

Further, the integrin alpha10 and/or alpha11 have several uses. Examplesof uses may thus be to identify, differentiate, and isolate mammalianmesenchymal stem cells from a mixed cell population as useful tools incell therapy to repair damaged tissue.

According to the invention, use of the integrin alpha10 and/or alpha11according to the invention is disclosed for identification of MSC.

Further, a use of the integrin alpha10 and/or alpha11 according to theinvention is disclosed, for modulating differentiation of a MSC.

Still even further, a use of the integrin alpha10 and/or alpha11according to the invention is disclosed, for isolating a MSC or anenriched population of MSC.

Mammalian MSC

In the methods and uses disclosed in the present invention, mammalianMSC, mammalian cellular populations and mammalian cellular compositionsare disclosed.

In specific embodiments, the mammal may be a human.

Still further embodiments include wherein the mammal is a rodent, suchas a rat, mouse, or any other member of the family Muridae.

EXAMPLES Example 1 Detection of Integrin Alpha10 and Integrin Alpha11Chain on Human MSC

Objective

The objective of this example is to analyse human MSC for the expressionof integrin alpha10 and alpha11, using immunoprecipitation.

Materials and Methods

Human mesenchymal stem cells (obtained from In Vitro, Sweden, at passage2), were cultured in MSCBM medium (provided by In Vitro, Sweden) untilpassage 4 and then surface biotinylated.

In brief, cells adherent on the plate were washed once with PBS and thensurface biotinylated using 0.5 mg/ml Sulfo-NHS-LC-biotin (Pierce) in 4ml PBS for 20 min. Cells were then washed once with PBS and 10 ml 0.1Mglycine/PBS were added for 5 min.

After washing once with PBS cells were lysed in 1 ml lysis buffer (1%NP40, 10% glycerol, 20 mM Tris/HCl, 150 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂,protease inhibitor cocktail BM, pH7.5). The cell lysate was collectedwith a plastic scraper, pipetted into an eppendorf tube and spun downfor 10 min at 15.000 g.

The supernatant collected from the centrifugation step above wasincubated with 2 microliter of alpha10 pre-immune serum followed byaddition of 20 μl Prot G sepharose (Amersham) in 100 μl lysis buffer.

After rotating the cells in lysis buffer over night at 4° C. the lysatewas centrifuged for 1 min at 8000 rpm and the supernatant collected. Foreach subsequent immunoprecipitation 150 μl cell lysate were pipettedinto an eppendorf tube and 1 μl of antiserum was added. The sera usedwere rabbit-anti-human al 0 and rabbit-anti-human all, respectively(both sera against the cytoplasmic domains of the integrins).

After 1 h rotating at 4° C., 20 μl protein G Sepharose (Amersham) in 100μl lysis buffer was added and the mixture further rotated for another 30min. The Sepharose-beads were then spun down briefly and washed threetimes with lysis buffer.

20 μl SDS-PAGE sample buffer (including 100 mM DTT) was added to theSepharose beads and then the samples were boiled for 5 min. 5 μl of eachsample were run on a 8% SDS-PAGE gel (Novex) and thenelectro-transferred onto a PVDF membrane.

The membrane was blocked in 2% BSA/TBST (TBST: 20 mM Tris/HCl pH 7.5,150 mM NaCl, 0.05% Tween 20) for 1 h, washed once with TBST and thenincubated with 2 μl Extravidin-peroxidase (Sigma) in 8 ml blockingbuffer.

After 1 h the Extravidin-peroxidase solution was removed and themembrane washed 3×20 min in TBST. Surface biotinylated proteins werethen detected with ECL (Amersham) and visualised on a photographic film.

Results and Discussion

In FIG. 2, the result of the immunoprecipitation is shown. Humanmesenchymal stem cells in culture express both integrins alpha10 andalpha11 on their surface. In the figure, the upper band is alpha10 (inthe left lane) and alpha11 (in the right lane). The lower band in bothlanes represents the beta1 chain. Both integrin alpha10 and alpha11expression is identified.

Example 2 Identification of HEK293 Cells Expressing the IntegrinAlpha10Beta1 and the alpha11beta1

Objective

The objective with this example is to use antibodies to alpha10 andalpha11 to identify and differentiate between HEK293 cells expressingthe integrin alpha10beta1 and the integrin alpha11beta1.

Materials and Methods

Integrin alpha10 and alpha11-transfected HEK293 cells andnon-transfected HEK293 cells were trypsinized, washed with PBS and thenincubated for 20 min with integrin antibodies against alpha10 andalpha11 (1 μg/ml in PBS supplemented with 1% BSA).

Labelled cells were washed twice with PBS/1% BSA and then incubated for20 min with PE labelled goat-anti-mouse Ig (Pharmingen, BD Biosciences)at a concentration of 1 μg/ml in PBS/1% BSA.

Cells were thereafter washed twice in PBS/1% BSA and were analysed on aFACSort® (Becton-Dickinson) by collecting 10,000 events with the CellQuest® software program (Becton-Dickinson).

Results

The results are shown in FIG. 3A-I as histograms after FACS-analysis.

In FACS assay, the antibody against alpha10 bound to the HEK293 cellstransfected with human alpha10 integrin-subunit, shown in FIG. 3E. Thiswas seen as a displacement in the FACS histogram to the right.

The antibody against alpha10 did not bind to HEK293 cells transfectedwith human alpha11 integrin-subunit, as shown in the middle panel(right, FIG. 3F), or untransfected HEK293 cells, as shown in FIG. 3D).

Similarly, the antibody against alpha11 bound to the HEK293 cellstransfected with human alpha11 integrin-subunit, shown in FIG. 3I. Thiswas seen as a displacement in the FACS histogram to the right. Theantibody against alpha11 did not bind to HEK293 cells transfected withhuman alpha10 integrin-subunit, as shown in FIG. 3H, or untransfectedHEK293 cells, as shown in FIG. 3G).

FIG. 3A-C represent control (secondary antibody alone), which did notbind to any of the HEK293 cells tested.

In summary FIG. 3A-I shows that HEK293cells expressing the integrinalpha10beta1 and integrin alpha11beta1 can be specifically sorted byFACS analysis using antibodies directed against these integrins.

Example 3 Identification of MSC Expressing the Integrin Alpha10 fromHuman Colonyforming Cells Derived from Human Bone Marrow

Objective

To test whether colony-forming cells derived from human bone marrowexpress the integrin alpha10 and represent a population of mesenchymalstem cells.

Materials and Methods

Human mononuclear bone marrow cells were isolated from bone marrow bydensity centrifugation.

30×10⁶ cells were taken in 20 ml medium (MSCGM medium provided byPoietics and delivered via Invitro, comprising 440 ml MSCBM (lot 017190)and 2×25 ml MCGS (lot 082295) and L-glutamine andPenicillin/Streptomycin) into a T75 flask and incubated in the cellincubator.

Cells were grown until day 12 (medium was changed twice) and thentrypsinized and split (5000 cells/cm²). In general, cells were split at90% confluency.

Cells were grown for a further 3 days and then split again (5000cells/cm²). At this point the influence of FGF-2 on alpha10-expressionon hMSC was investigated: One plate was grown in medium as before andone plate was grown in medium+5 ng/ml FGF-2. After two weeks in culturewith or without FGF-2 (including one passage) the cells were analysed byFACS using a monoclonal antibody to alpha10 as a means to analyse thecells.

Results

The results are shown in FIG. 4 as flow cytometry histograms.

After 2 weeks treatment with FGF-2, 96% of the cells treated with FGF-2expressed the integrin alpha10 (lower panel, FIG. 4 b).

Control (secondary antibody alone) is shown in the upper panel in FIG. 4a. Cells were tested for non MSC markers (CD34 and CD45) and found to benegative (results not shown).

Cells were also analysed for their morphology and light microscopyrevealed that the cells formed colonies typical of MSCs.

1. A method for producing an isolated population of mammalian cellsenriched for mesenchymal stem cells relative to a reference population,the method comprising the steps of a) providing at least a portion of apopulation of cells, or a portion of a reference population, comprisingat least one mesenchymal stem cell and at least one cell other than amesenchymal stem cell, b) introducing into the population of cells in a)above an antibody or fragment thereof identifying an integrin alpha 10chain expressed on the cell surface of mesenchymal stem cells, c)selecting and isolating from the population of cells in b) above themesenchymal stem cell(s) expressing integrin alpha 10 chain, therebyproducing a population of cells enriched for mesenchymal stem cells. 2.The method according to claim 1, wherein the mesenchymal stem cells areidentified as mesenchymal stem cells by detecting expression of integrinalpha 10 chain on the cell surface of said mesenchymal stem cells. 3.The method according to claim 1, wherein the selection in c) above isperformed by fluorescent cell sorting.
 4. A method for isolatingmammalian mesenchymal stem cells comprising the steps of a) providing acell suspension comprising mammalian mesenchymal stem cells and at leastone cell other than a mesenchymal stem cell, b) contacting the cellsuspension with an antibody or fragments thereof binding to integrinalpha10 chain expressed on the cell surface of mesenchymal stem cells,c) separating cells binding to said antibody or fragments thereof in b),and optionally d) recovering the cells binding to the antibody orfragments thereof in c) from said antibody or fragments thereof, therebyproducing a population of isolated mammalian mesenchymal stem cells,optionally free from said antibody or fragments thereof.
 5. The methodof claim 1, wherein the selection in c) above is performed using a solidsupport.